Loss of primary sensory neurons in the very old rat Neuron number estimates using the disector method and confocal optical sectioningкод для вставкиСкачать
THE JOURNAL OF COMPARATIVE NEUROLOGY 396:211–222 (1998) Loss of Primary Sensory Neurons in the Very Old Rat: Neuron Number Estimates Using the Disector Method and Confocal Optical Sectioning E. BERGMAN* AND B. ULFHAKE Department of Neuroscience, Karolinska Institute, S-171 77 Stockholm, Sweden ABSTRACT Loss of neurons has been considered to be a prime cause of nervous disturbances that occur with advancing age. However, the notion of a constitutive aging-related loss of neurons has been challenged recently in several studies that used up-to-date methods for counting neurons. In this study, we have applied stereological techniques with the objective of obtaining quantitative data on total neuron numbers and the distribution of neuron cross-sectional areas in the fifth cervical (C5) and fourth lumbar (L4) dorsal root ganglion (DRG) of 3- and 30-month-old Sprague-Dawley rats. Tissue data were recorded on a confocal laser-scanning microscope with the use of the optical-disector technique and random, systematic sampling. Aged rats of both sexes disclosed only a small decrease (<12%) in the number of cervical and lumbar DRG neurons. Furthermore, there was no significant correlation between the degree of neuron loss and the extent of behavioral deficits among the aged individuals. The DRG neurons of aged rats had a smaller mean cross-sectional area (<15%; P , 0.001) at both DRG levels. Further analysis of the male cohorts was carried out by using isolectin B4 and neurofilament subunit (phosphorylated 200 kDa; RT97) immunoreactivity (IR) as selective markers for unmyelinated and myelinated axons, respectively, and disclosed no significant change in the relative frequencies of immunoreactive neuron profiles in the old rats. However, RT97-IR DRG neurons of the aged rats had significantly smaller cross-sectional areas (<9% in C5; <16% in L4; P , 0.001) than the young adult rats, indicating a selective cell body atrophy among myelinated primary afferents during aging. The results indicate that loss of primary sensory neurons cannot exclusively explain the functional deficits in sensory perception among senescent individuals. It seems likely that other factors at the subcellular level and/or target interaction(s) contribute substantially to the sensory impairments observed with advancing age. J. Comp. Neurol. 396:211–222, 1998. r 1998 Wiley-Liss, Inc. Indexing terms: aging; stereology; optical disector; dorsal root ganglia; spinal cord Aging is associated with neurological symptoms and signs that are suggestive of peripheral neuropathy (Critchley, 1956; Cavanagh, 1964; Jellinger, 1973; Kokmen et al., 1977). Thus, increased thresholds for tactile, thermal, and vibratory sensations are common in senescent mammals (Dyck et al., 1984; de Neeling et al., 1994; Gescheider et al., 1994; Bergman et al., unpublished observations), and studies of sensory nerves in aged animals have shown axonal dystrophy, demyelination, and axon degeneration (Berg et al., 1962; van Steenis and Kroes, 1971; Gilmore, 1972; Burek et al., 1976; Thomas et al., 1980; CotardBartley et al., 1981; Mitsumori et al., 1981; Mittal and Logmani, 1987). Loss of neurons during senescence, which has long been considered to be an important trait of aging, is currently r 1998 WILEY-LISS, INC. the subject of reexamination (for review, see Wickelgren, 1996). Earlier studies of neuron loss during aging were often compromised by inconsistent results (for review, see Coleman and Flood, 1987). For example, results from Grant sponsor: Swedish Medical Research Council; Grant number: 10820; Grant sponsor: Karolinska Institutet; Grant sponsor: L and H Ostermans Fond för Medicinsk Forskning; Grant sponsor: A and M Wallenbergs Minnesfond; Grant sponsor: Kapten A. Erikssons Stiftelse för Medicinsk Forskning. *Correspondence to: Esbjörn Bergman, Department of Neuroscience, Karolinska Institute, S-171 77 Stockholm, Sweden. E-mail: email@example.com Received 6 August 1997; Revised 29 December 1997; Accepted 1 January 1998 212 E. BERGMAN AND B. ULFHAKE studies of dorsal root ganglia (DRGs) are at variance, with some revealing a loss of up to one-third of the neurons with advancing age (Gardner, 1940; Nagashima and Oota, 1974), whereas others show no reduction in cell numbers (Emery and Singhal, 1973; Ohta et al., 1974; La Forte et al., 1991). A problem with all of these studies is that they were performed by using indirect techniques based on two-dimensional images or probes, thereby producing biased results (e.g., see Coggeshall, 1992). With the development of stereological techniques (for reviews, see Sterio, 1984; Gundersen et al., 1988a,b), it seems possible to circumvent these methodological problems in producing accurate estimates of neuron numbers. Since a precise knowledge about the morphometric characteristics of DRGs may help us to understand some aspects of the functional deficits occurring with advancing age, we have applied stereological techniques to estimate the total number of neurons and their cross-sectional areas in cervical and lumbar DRGs of young adult (3 months old) and aged (30 months old) Sprague-Dawley rats. Both males and females were included to examine any possible difference(s) related to sex. Furthermore, a distribution analysis, with immunoreactivity for the phosphorylated 200-kDa neurofilament subunit (RT97) and isolectin B4 as markers for large light (myelinated; Lawson et al., 1984) and small dark (unmyelinated; Wang et al., 1994) DRG neurons, respectively, was performed in order to shed some light on any possible selectivity of aging on subpopulations of DRG neurons. MATERIALS AND METHODS Experimental animals and behavioral deficits in aged rats In this study, male and female Sprague-Dawley rats (strain Bkl; Harlan Sprague-Dawley, Houston, TX), including a total of nine young adult (2–3 months old; body weight: male 300–400 g, n 5 4; female 200–250 g, n 5 5) and eight aged rats (30 months old; body weight: male 590–700 g, n 5 3; female 290–400 g, n 5 5), were used. The animals were delivered by a local breeder (B and K, Stockholm, Sweden) at 2 months of age and were kept thereafter under standardized barrier-breeding conditions at our department (12-hour light/12-hour dark cycle) with free access to water and food (R70 with reduced protein content; Lactamin, Vadstena, Sweden). Under these conditions, the median life span is about 30 months (62 months across cohorts) for both males and females (see also Gutman and Hanzlikova, 1972; Burek and Hollander, 1980; Masoro, 1980; Algeri et al., 1983). Based on this, the 30-month-old rats were defined as ‘‘aged.’’ Rats show a progressive deterioration of motor behavior during aging (Berg et al., 1962; van Steenis and Kroes, 1971; Burek et al., 1976; Mitsumori et al., 1981; Krinke, 1983; Johnson et al., 1993), with symptoms usually starting during the third year of life and affecting mainly the hindlimbs (‘‘posterior paralysis’’). All aged rats used in this study disclosed signs of behavioral sensory-motor disturbances, defined according to a previously described staging protocol (Johnson et al., 1995). Briefly, the symptoms were most evident in the hindlimbs and ranged from a moderate muscular atrophy and an adduction insufficiency in the least affected cases (stage I) to a more or less complete paralysis of the hindlimbs with severe wasting of the hindlimb muscles in the most severely affected animals (stage III). Among the aged rats used in this study, all three stages were represented. Behavioral testing of sensory functions included nociceptive (hot-plate test; Espejo and Mir, 1993; Langerman et al., 1995) and tactile (von Frey test; Tal and Bennett, 1994) thresholds and will be described in detail elsewhere (Bergman et al., unpublished observations). Briefly, nociceptive thresholds (response latency) were increased in aged rats (see Fig. 1A), and there was a positive correlation between response latency and age/stage (Fig. 1A). Also, tactile thresholds increased significantly from 3 month to 30 months of age (only females tested; Fig. 1B). In the latter test, however, no consistent correlation to clinical stage could be revealed among the aged rats. All experiments were approved of by the Local Ethical Committee (Stockholm’s Norra Djurförsöksetiska Nämnd; project no. 263/ 95). General procedures The rats were deeply anaesthetized with chloral hydrate (300 mg kg21, i.p.) and perfused transcardially with warm (37°C), Ca21-free Tyrode’s solution followed by cold fixative (4°C) containing 4% w/v paraformaldehyde and 0.2% w/v picric acid in 0.1 M phosphate-buffered saline (PBS), pH 7.2; Pease, 1962; Zamboni and De Martino, 1967) for 8 minutes. The fifth cervical (C5) and fourth lumbar (L4) DRGs were dissected out, immersed in fresh fixative for 90 minutes, and then stored in 10% sucrose buffer solution containing 0.01% sodium azide (Sigma, St. Louis, MO) and 0.02% Bacitracin (Bayer, Leverkusen, Germany) overnight at 4°C. Complete section series of the DRGs randomly orientated around their longitudinal axis were cut at 14 µm in a cryostat (Dittes, Heidelberg, Germany) and thaw mounted onto chrome-alum gelatin-coated slides. Tissue preparation Conventional histochemical procedures. With a random starting point, sections to be used for cell counting were systematically sampled throughout the complete section series and stained with a fluorescent counterstain. Briefly, the air-dried sections were rehydrated in 0.1 M PBS, counterstained for 5 minutes in 0.001% (w/v) propidium iodide (Sigma) in PBS, rinsed three times for 5 minutes each in PBS, and mounted in glycerol. Propidium iodide, which is known to bind to nucleic acids, yielded a pattern that closely resembled conventional Nissl staining, with fluorescent brightly cytoplasm and nucleoli. Immunohistochemical procedures. Remaining sections from male young adult and aged rats were used for B4 or RT97 immunohistochemistry according to the indirect immunofluorescence technique of Coons and collaborators (see Coons, 1958). After rehydration in 0.1 M PBS, the sections were preincubated for 1 hour at room temperature in PBS containing 10% normal donkey serum and 0.3% Triton X-100. For B4 immunohistochemistry, sections were incubated with the isolectin B4 from Griffonia simplicifolia I (B4; 10 µg/ml; Vector Laboratories, Burlingame, CA) for 18 hours at 4°C. The sections were then rinsed three times for 10 minutes each in PBS and incubated with goat antiGriffonia simplicifolia lectin I (GSA I) antiserum (1:1,000; Vector Laboratories) for 18 hours at 4°C. For RT97 immunohistochemistry, sections were incubated with a mouse anti-RT97 monoclonal antibody (phosphorylated 200 kDa NEURON LOSS IN AGED RAT DRG 213 Fig. 1. Effects of aging on nociceptive (hot-plate test; A) and tactile (von Frey test; B) thresholds. The aged rats (stages I–III) disclosed significantly higher thresholds for both nociceptive and tactile stimuli than the young adult rats (analysis of variance [ANOVA], P , 0.001). In the hot-plate test, there was a positive correlation between response latency and age/stage (Spearman correlation coefficient 5 0.59; P , 0.001), whereas, in the von Frey test, no such correlation was found among the aged rats. Each point represents the mean 6 standard error of mean (S.E.M.). Levels of significance are described in Materials and Methods under Statistics. neurofilament (1:250, kindly provided by J.W. Wood; Wood and Anderton, 1981) for 72 hours at 4°C. After incubation with the primary antibodies, the sections were thoroughly rinsed in PBS and incubated with dichlorotriazinyl aminofluorescein (DTAF)-conjugated donkey anti-goat IgG (1:40; Jackson Immuno Research, West Grove, PA) or DTAF-conjugated donkey anti-mouse IgG (1:40; Jackson Immuno Research) for 30 minutes at 37°C. After a final rinse in PBS, the sections were mounted in glycerol/PBS (3:1) containing 0.1% r-phenylenediamine in order to retard fading (Johnson and de C Nogueira Araujo, 1981; Platt and Michael, 1983). All antisera/antibodies were diluted in 0.1 M PBS containing 1% bovine serum albumin, 0.3% Triton X-100, 0.01% sodium azide, and 0.02% Bacitracin. DRG volume. The Cavalieri method (Gundersen and Jensen, 1987) was used to estimate the volume of the DRG. With a random starting point, 10–15 equally spaced sections through the ganglia were analyzed. Each section was recorded with a 43 objective (5-µm pixel spacing), and the cross-sectional area of each ganglion section was measured by using Image Space software (Fig. 2A). For measurements of section thickness, axial (z-)-scans (0.10-µm step size) were sampled in a random systematic fashion across each section with a 403 objective (Fig. 2B). With the used rehydration and embedding protocol (see above), the tissue shrinkage was negligible (Fig. 2B). Tests with ethanol dehydration and embedding in Entellan (Merck, Darmstadt, Germany) resulted in a 30–50% shrinkage of the tissue sections (data not shown). The total ganglion volume was calculated by multiplying the mean cross-sectional area, the mean section thickness, and the number of sections. Neuron density. The optical-disector principle (Gundersen, 1986; Gundersen et al., 1988a) was applied on the sections used for the Cavalieri estimates. The sampling scheme employed was based on the results from a pilot study in which the appropriate number of disectors was determined. In total, approximately 65 disectors were analyzed in each ganglion. With a random starting point in each section, visual fields were sampled systematically by using an x-y-axis step size of 600 µm or 800 µm for the fifth cervical vertebrae (C5) and the fourth lumbar vertebrae (L4) DRGs, respectively. At each sample location, optical sectioning was performed with the CLSM by using a 403/1.0 NA planApo oil-immersion objective with an axial resolution of 2.5 µm (defined by the full-width half maximum; Stereology To obtain an unbiased estimate of the total number of neurons, we used the disector principle and random systematic sampling (Sterio, 1984; Gundersen et al., 1988a; West, 1993). The analysis was performed on a Sarastro 1000 (Molecular Dynamics, Inc., Sunnyvale, CA) confocal laserscanning microscope (CLSM) with laser wave lengths and filters set for propidium iodide fluorescence. Briefly, the 514-nm line of an argon-ion laser was used as the excitation light, and the propidium iodide fluorescence emission light was collected through a dichroic mirror (split wave length 525 nm) and a long-pass filter (LP 530 nm). Images were recorded with either a 403/1.0 oil-immersion planapochromate objective or a 43 air objective and stored in a computer for subsequent analysis in Image Space software (Molecular Dynamics, Inc.). 214 E. BERGMAN AND B. ULFHAKE Figure 2 NEURON LOSS IN AGED RAT DRG see Ulfhake et al., 1994). The starting point, which was derived from a z-scan, was set 2 µm below the section surface. From this point, nine consecutive optical sections were recorded by using a z-axis step size of 1 µm. The resulting stack of digital images, with a lateral pixel spacing of 0.25 µm, was analyzed on a computer equipped with the Image Space software. An unbiased counting frame with an area of 39,828 µm2 was presented by the computer, and all neurons with a nucleolus in the starting plane (i.e., optical section one) or in the guard volume, which includes the immediately adjacent mechanical section, were disregarded. In the following eight sections, i.e., section two through nine, all neurons with a distinct nucleolus appearing inside the counting frame were counted (Fig. 2C–K). In this way, by using optical disectors with a height of 8 µm, an average of 165 neurons were sampled in each ganglion. The neuron density was then calculated as the sum of all neurons counted divided by the summed volume of all disectors. Neuron numbers and cross-sectional areas. The total number of neurons was calculated as the product of the volume and the numerical density, i.e., the number of neurons per unit volume, in each ganglion. All cells sampled during the stereological procedure had their cross-sectional area measured in the plane of the nucleolus. The measurements were performed on the digitized images, and the results were stored in the computer for subsequent analysis. Quantitative evaluation of B4- and RT97-immunoreactive neuron profiles To determine the relative frequencies and size distributions of B4- and RT97-immunoreactive (IR) neuron profiles in C5 and L4 DRGs, eight to ten sections were sampled systematically throughout each ganglion in both young adult and aged male rats. In each section, two to four randomly selected fields were collected by using a Nikon microscope (Tokyo, Japan), with a 203/0.75 NA objective lens equipped with a Ultrapix 1600 CCD camera (AstroCam Ltd., Cambridge, United Kingdom). The digital images, each comprising 1,536 3 1,024 pixels with 8 bits of Fig. 2. A–K: Confocal images illustrating the principles of the optical disector method used for quantification of total neuron numbers and cross-sectional areas. A: Overview, which was scanned with a 43 objective, of a randomly, systematically sampled section in a fourth lumbar vertebrae (L4) dorsal root ganglion (DRG) of a young adult male rat. Within each selected section, a random starting point was chosen, and fields were then sampled systematically and analyzed (squared area in A). The overview image is also used to measure the cross-sectional area of the DRG section for the estimation of total ganglion volume, according to the Cavalieri principle. B: A z-scan, which was captured with a 403/1.0 NA plan-Apo oil-immersion objective, revealing a section thickness of 14 µm. C–K: Confocal optical sections, which were scanned with a 403/1.0 NA plan-Apo oilimmersion objective, through the tissue specimen. Starting 2–3 µm below the section surface, as decided from the preceding z-scan, nine consecutive images were recorded by using a step size of 1 µm. All neurons containing a nucleolus in the first section (arrowheads in C) were disregarded. In the following eight sections (D–K), all neurons with a distinct nucleolus (solid arrows in I and K) that appeared within the unbiased counting frame (square in C–K) were counted and had their cross-sectional area measured. Nucleoli touching the upper or right border of the counting frame were considered to be inside, whereas those touching the lower or left border (open arrow in I) were considered to be outside and, hence, were not counted. Scale bars 5 200 µm in A, 5 µm in B, 20 µm in C (also applies to D–K). 215 data, were analyzed by using the Optimas 6.0 software (Optimas Corporation, Bothell, WA) to calculate the ratios of labeled/unlabeled neuron profiles. Only cell profiles containing a clearly visible nucleus were included. All immunopositive neuron profiles also had their crosssectional area measured in the nuclear plane. In total, 830–1,280 profiles were analyzed in each DRG. Statistics To analyze the appropriateness of the sampling scheme employed, the coefficient of variance (CV; S.D./mean) and the coefficient of error (CE) were determined. The CE of the individual estimates, reflecting the precision of the sampling procedure, was calculated according to West and Gundersen (1990; see also Gundersen and Jensen, 1987). Analysis of variance (ANOVA) with Fisher’s LSD was used 1) to test for the effects of age and sex, respectively, on total neuron numbers at the two DRG levels studied; and 2) to evaluate the effect of aging on the relative frequency and size distribution of neuron profiles immunoreactive for B4 and RT97 in C5 and L4 DRGs. When analyzing the cell-size distribution, nonparametric Kruskal-Wallis one-way ANOVA and contingency table analysis (x2; bin data) were used. For contingency table analysis, each set of data was divided into three size bins corresponding to small (,750 µm2), medium (750–1,750 µm2), and large (.1,750 µm2) DRG neurons, respectively (see also Rambourg et al., 1983; Price, 1985; Tandrup, 1993; Zhang et al., 1994). In the histograms, the levels of significance are been indicated as follows: n.s., nonsignificant; single asterisk, P , 0.05; double asterisks, P , 0.01; triple asterisks, P , 0.001 (see Figs. 1, 3). RESULTS Numbers of neurons The numbers of neurons in C5 and L4 DRGs of young adult and aged rats of both sexes are indicated in Figure 3. Aged animals, on the average, had 12% fewer cells in both C5 (11.7–13.9%) and L4 (10.8–13.5%) DRGs, with no difference in the degree of cell loss between male and female rats. The decrease in neuron numbers with age was highly significant for female L4 DRGs (ANOVA; P , 0.001) and, to a lesser extent, also for cervical ganglia of both sexes (ANOVA; P 5 0.04 and P 5 0.02 for male and female rats, respectively). The small difference between young adult and aged male L4 DRGs was not significant (ANOVA; P 5 0.11). No correlation was found between the degree of cell loss and the clinical symptoms (stage) of the rats (Fig. 4). Both young adult and aged male rats showed approximately 6% fewer cells than female rats in both cervical and lumbar DRGs. However, these differences were not statistically significant (ANOVA; P 5 0.09–0.26). Aged rats, of both sexes, had an increased DRG volume of approximately 16% (C5) and 26% (L4). In parallel, a consistent reduction in the neuron density was observed. Analysis of the sampling scheme revealed that the mean observed relative variation among animals, coefficient of variation (CV2), was 0.0087, and the average coefficient of error (CE2; see Materials and Methods) of the estimates was 0.0024, with an equal contribution of the estimates for ganglion volume and numerical density. Thus, the biological variance contributed more than two-thirds of the total variance. 216 E. BERGMAN AND B. ULFHAKE Fig. 3. Graphic representation of the total number of neurons in the fifth cervical (C5) and fourth lumbar (L4) dorsal root ganglia (DRG) in young adult (open symbols) and aged (solid symbols), male (squares) and female (circles), Sprague-Dawley rats. Horizontal bars represent mean values. Levels of significance are described in Materials and Methods under Statistics. Fig. 4. Graphic representation of the relation between the total number of neurons in C5 (below hatched line) and L4 (above hatched line) dorsal root ganglia (DRG) and age (young adult, open symbols; aged stages I–III, solid symbols). Male and female rats are indicated with squares and circles, respectively. Note the lack of correlation between neuron loss and symptoms among the aged rats at both DRG levels studied. TABLE 1. Mean Cross-Sectional Areas in C5 and L4 DRGs of Young Adult (Female and Male) and Aged (Female and Male) Rats1 C5 cross-sectional area µm2 (mean 6 S.D.) L4 cross-sectional area µm2 (mean 6 S.D.) Cell size distribution Cell body cross-sectional areas in cervical and lumbar DRGs of both female and male aged rats were found to be approximately 15% smaller than in young adult rats (Table 1, Fig. 5). The difference in cell size distribution between young adult and aged rats was statistically significant (Kruskal-Wallis; P , 0.001) in both C5 and L4 DRGs. Comparison of male and female rats within age groups revealed no statistically significant difference (Kruskal-Wallis; P 5 0.06–0.57). Figure 5 also illustrates the frequency of neurons in the three size categories used to define small, medium, and large DRG neurons. The histograms clearly indicate a shift toward smaller cell categories among the neuron populations of aged animals. A significant difference was evident in both C5 and L4 DRGs when comparing young adult and aged rats (x2; P , 0.001) of both sexes, whereas no difference was seen between males and females within the age groups (x2; P 5 0.08–0.55). Examination of B4- and RT97-IR neurons The relative distribution of B4 and RT97 immunoreactivity (Fig. 6) was examined in the male age cohorts to 1C 5, Young adult (n 5 9) Aged (n 5 8) 926 6 592 1,165 6 809 784 6 472 993 6 614 fifth cervical vertebrae; L4 , fourth lumbar vertebrae; DRGs, dorsal root ganglia. determine whether the observed reduction in cell-number and cell-size in aged rats, showed any selectivity for non-myelinated or myelinated DRG neurons. In Table 2, the relative frequencies of B4- and RT97-IR profiles in young adult and aged rat DRGs have been tabulated. The data show that the relative proportions of the two neuron populations at both DRG levels remain virtually unchanged during aging (ANOVA; P 5 0.27–0.94). Examination of the distribution of cross-sectional areas of B4-IR neuron profiles showed that the mean area in the aged rats was approximately 2% smaller than in the young adult rats in both C5 and L4 DRGs. However, among RT97-IR neuron profiles, 9% (ANOVA; P , 0.001) and 16% (ANOVA; P , 0.001) decreases in mean cross-sectional area were found in C5 and L4 DRGs, respectively, of the aged rats. The difference was most prominent for L4 DRGs, in which all aged rats were significantly different from the young adult rats, whereas, in C5 DRGs, the aged rats differed significantly from two of the young adult rats. Thus, cell body atrophy in aged rats was only recorded in the myelinated subpopulation of DRG neurons. NEURON LOSS IN AGED RAT DRG 217 Fig. 5. Size frequency histograms illustrating the distribution of C5 (A) and L4 (B) dorsal root ganglia (DRG) neurons in young adult (male 1 female; gray) and aged (male 1 female; black) rats. The frequency (6S.D.) of neurons that belong to each of the three size bins (indicated by vertical hatched lines), as defined in Materials and Methods, is indicated for both age groups. Note the shift toward smaller cells among the aged rats. DISCUSSION Changes in cell body cross-sectional areas seem to implicate a selective atrophy of large myelinated primary afferents during aging (see also below). However, our data do not suggest that neuron loss in DRGs was more extensive among any subpopulation of primary afferents. This study shows that aging is associated with only a small loss of DRG neurons, equally evident at cervical and lumbar levels, in Sprague-Dawley rats of both sexes. 218 E. BERGMAN AND B. ULFHAKE Fig. 6. Immunofluorescence images of young adult (A,C) and aged (B,D) male rat L4 dorsal root ganglia (DRGs) after incubation with B4 (A,B) and RT97 (C,D). With regard to frequencies of immunopositive neuronal profiles, no difference could be observed when comparing young adult and aged rats. However, for RT97, but not for B4, a significant decrease in mean cross-sectional area could be observed in the aged compared with the young adult rats. Small (A,B), medium (A–D), and large (C,D) arrows point to small, medium-sized, and large DRG neurons, respectively. Scale bar 5 70 µm. TABLE 2. Frequencies and Mean Cross-Sectional Areas of B4- and RT97Immunoreactive neurons in C5 and L4 DRGs of Young Adult and Aged Rats and confocal microscopy. In contrast to conventional microscopy, confocal microscopy provides a resolution along the optical axis (for references, see Ulfhake et al., 1994), enabling true optical sectioning of tissue specimens. Thus, recording of profiles in focus is operator independent and is therefore unbiased. We have chosen to count a neuron the first time that a nucleolus appears in the profile, which is a unique event. Quite frequently, multiple nucleoli were encountered, especially in the smaller type-B cells. To secure the validity of the counting, the tissue volume above the starting plane (i.e., guard volume), including the immediately adjacent mechanical section, was checked for possible occurence of multiple nuclei/nucleoli in the counted cell. Even though the methodology employed here is considered insensitive to tissue shrinkage with regard to total neuron numbers, shrinkage will affect the crosssectional area measurements and, moreover, will increase the demand on the optical depth resolution. To circumvent the problem with tissue shrinkage, observed in, e.g., paraffin sections (see Schmalbruch, 1987), the sections were rehydrated in PBS and embedded in glycerol, which preserves the tissue volume. In test experiments with tissue embedding in Entellan (Merck) following dehydra- C5 Young adult (n 5 4) B4 Frequency (% 6 S.D.) Cross-sectional area µm2 (mean 6 S.D.) RT 97 Frequency (% 6 S.D.) Cross-sectional area µm2 (mean 6 S.D.) L4 Aged (n 5 3) Young adult (n 5 4) Aged (n 5 3) 51 6 7 51 6 5 50 6 5 50 6 7 612 6 157 587 6 131 726 6 199 718 6 181 43 6 7 44 6 8 44 6 8 46 6 7 1,384 6 467 1,266 6 412 1,923 6 590 1,612 6 511 Furthermore, there were no indications of a correlation between neuron loss and the degree of clinically manifest dysfunction among the aged rats. Methodological considerations To our knowledge, this is the first attempt to quantify changes in DRGs of both sexes during aging by using stereological techniques. Here, we have employed the disector principle (Sterio, 1984; Gundersen et al., 1988a) NEURON LOSS IN AGED RAT DRG tion, we recorded a reduction of the section thickness by approximately 30–50%. It was calculated that the biological variance contributed more than two-thirds of the total variance in each age group and spinal cord level, which is satisfactory, in that the stereological approach contributes only a minor fraction of the observed variance. Number of DRG neurons and loss of neurons during aging Our values for total number of L4 DRG neurons in young adult rats are close to those obtained with the same technique in rat L5 DRGs (Tandrup, 1993). Earlier estimates using other counting methods have shown a severalfold difference in neuron numbers across studies (Bondok and Sansone, 1984; Feringa et al., 1985; Arvidsson et al., 1986), and this inconsistency has been attributed to imperfection(s) in the applied counting procedures (Coggeshall et al., 1990; Pover and Coggeshall, 1991). Furthermore, with the exception of a complete serial section study of DRGs (Schmalbruch, 1987), neuron counts in the studies cited above have yielded much lower values than those obtained with the disector technique. Examination of neuron number in DRGs during aging has produced inconsistent results (Gardner, 1940; Emery and Singhal, 1973; Nagashima and Oota, 1974; Ohta et al., 1974; La Forte et al., 1991), as discussed above, and this variation is most likely related to the counting procedures used. The results obtained here show that loss of primary sensory neurons is small also in the very old rat and, furthermore, that there was no discernible difference in the degree of cell loss between sexes or between levels of the spinal cord. The findings implicate that cell loss itself cannot account for the sensory deficits seen in elderly individuals. Furthermore, our results are consistent with studies of dorsal roots that have described only small or no loss of (myelinated) fibers with advancing age (Mitsumori et al., 1981; Rao and Krinke, 1983; Knox et al., 1989). The present findings, moreover, add to the growing body of studies showing that aging is not accompanied by a substantial loss of neurons (see e.g., Satorre et al., 1985; Ahmad and Spear, 1993; Madeira et al., 1995; for reviews, see Coleman and Flood, 1987; Wickelgren, 1996). Aged rats In this study, the 30-month cohort was used as ‘‘aged.’’ In the literature, the median survival age of rats varies between 26 months and 32 months (Gutman and Hanzlikova, 1972; Burek and Hollander, 1980; Masoro, 1980; Algeri et al., 1983; Johnson et al., 1993, 1995; Hashizume and Kanda, 1995). Outbred Sprague-Dawley rats kept in a barrier facility, on average, reach an age of about 30 months, and, as described here and elsewhere, litter mates at this age disclose highly variable degrees of nervous function incapacitations, reflecting differences among the individuals (Bergman et al., unpublished observations; see also van Steenis and Kroes, 1971; Burek et al., 1976; Johnson et al., 1995). Although the number of DRG neurons varied among the aged rats, this variation did not appear to be linked to the extent of sensory-motor dysfunction of the individuals, to sex, or to the level of the spinal cord. 219 Sensory impairments and selective vulnerability during aging With advancing age in both rodents and humans, exteroception and proprioception become impaired (Foster et al., 1976; Macintosh and Sinclair, 1978; Kenney and Fowler, 1988; Schmidt et al., 1990; Ferrell et al., 1992; AbdelRahman and Cowen, 1993; de Neeling et al., 1994; Gescheider et al., 1994; Ferrer et al., 1995; Quoniam et al., 1995; Robbins et al., 1995). Peripheral nerves of aged animals show both loss of fibers and degenerative changes (Cowen et al., 1982; Dhall et al., 1986; Mione et al., 1988; Cowen and Thrasivoulou, 1990; Navarro and Kennedy, 1990; Abdel-Rahman and Cowen, 1993). These data are consistent with studies of sensory nerves and dorsal roots demonstrating axonal dystrophy, demyelination, and axon degeneration as well as loss of fibers during aging (Berg et al., 1962; van Steenis and Kroes, 1971; Gilmore, 1972; Burek et al., 1976; Sharma et al., 1980; Thomas et al., 1980; Cotard-Bartley et al., 1981; Mitsumori et al., 1981; Krinke, 1983; Mittal and Logmani, 1987; Knox et al., 1989). It seems that peripheral nerves are affected more than both dorsal roots (Rao and Krinke, 1983; Knox et al., 1989) and the parent cell bodies in the DRGs. A considerable loss of dermal innervation has been described in skin from both rodents and humans (see e.g., Abdel-Rahman and Cowen, 1993; Fundin et al., 1997), implicating poorer thermal and tactile sensibility. This is consistent with our behavioral tests of aged rats, which show increases in both hot-plate response latency and von Frey hair threshold. Furthermore, several studies have shown that there appear to be specific patterns for sensory fiber loss during aging (Cauna, 1965; Fundin et al., 1997), for example, with aging, proprioceptive receptors/sensory neurons degenerate earlier and more extensively than nociceptive fibers (Fundin et al., 1997). A differential effect of aging on tactile skin receptors has also been demonstrated in both rodents and humans (Cauna, 1965; Winkelmann, 1965; Bolton et al., 1966; Schimirgk and Ruttiger, 1980; Cerimele et al., 1990; Fundin et al., 1997). Furthermore, physiological tests of tactile responses in the finger pulp (Schmidt et al., 1990) have indicated that the distal axon, including the endings, seems to be affected more severely than the proximal axon, thus, supporting the notion that neuronal aging is often a distal-to-proximal process. Proprioceptive signals are conveyed most often in Aa and Ab fibers, whereas nociception travels in Ad and C fibers. Axon dystrophy, degeneration, and demyelination in peripheral nerves and spinal roots are particularly frequent in myelinated, large-diameter fibers (Spencer and Ochoa, 1981; Krinke, 1983; Rao and Krinke, 1983; Knox et al., 1989). The stereological data recorded in aged rat DRGs may be interpreted to show that aging is associated with a selective loss of large (myelinated) DRG neurons. However, it cannot be excluded that cell loss and cell atrophy occur as independent processes during aging and, hence, that cell loss may be unselective concerning cell size, whereas, compared with small DRG neurons, large DRG neurons may be more prone to atrophy. To shed some light on this issue, we employed immunohistochemistry by using B4 and RT97, respectively, as markers for unmyelinated and myelinated neurons. This examination revealed no difference between young adult and aged rats in relative frequencies of B4- and RT97-IR neuron profiles, 220 E. BERGMAN AND B. ULFHAKE indicating that there is probably no selective loss of small or large DRG neurons during aging. However, the RT97-IR cells, which presumably give rise to large myelinated axons, showed a pronounced reduction in mean crosssectional area in the nuclear plane. Thus, it may indeed be that the decrease in neuron size recorded in the stereological part of this study can be explained by a selective atrophy among RT97-IR neurons. The cell body atrophy among sensory neurons observed here is consistent with a previous report (Rao and Krinke, 1983) in which a decreased mean cell size was found in aged rat DRGs. Several studies have shown a decreased expression of neurofilaments in aged rat primary sensory neurons (Parhad et al., 1995; Kuchel et al., 1996), which is interesting, because neurofilaments are the major determinants of axonal caliber (Hoffmann et al., 1987). A decreased capacity for, in particular, large sensory neurons to sustain their cytoskeletal framework could be a plausible explanation for axon dystrophy/cell atrophy. The mechanism behind a decreased expression of neurofilaments remains unclear. However, the expression of neurofilaments is believed to be regulated by a neurotrophic signal from the periphery (Gold et al., 1991; Parhad et al., 1995), and we have shown previously that aged rats have a decreased expression of neurotrophin receptors (Bergman et al., 1996). Clearly, this issue cannot be resolved here but deserves further study. In this context, it seems appropriate also to comment on the notion that neuropathy in the elderly is caused commonly by mechanical trauma, like nerve entrapment or disc hernia. However, the characteristic changes in neurons with advancing age are common in the mystacial pad (Fundin et al., 1997), the nerve to the tail (Thomas et al., 1980), and systems intrinsic to the central nervous system (Johnson et al., 1993); furthermore, they also show a high selectivity within a nerve, making it highly unlikely that pressure trauma alone is the common etiology to neuropathy among senescent individuals. CONCLUSIONS Loss of neurons has long been considered a key element contributing to the symptomatology observed in elderly individuals. Our results using confocal microscopy and the disector technique show that the degree of DRG neuron loss in aged rats is at a level that is unlikely to explain fully the functional deficits. Furthermore, the reduction in neuron numbers did not differ between spinal cord levels or between sexes; moreover, there was a lack of correlation between behavioral symptoms and neuron loss among the aged rats. 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